1. Field of Invention
The present invention relates to the microbiological industry, and specifically to a method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family, wherein the glycogen biosynthesis pathway has been disrupted.
2. Description of the Related Art
Glycogen represents the major form of stored carbon for Escherichia coli and many other prokaryotes, and provides a readily metabolized substrate for maintenance energy. Glycogen accumulation in Escherichia coli is inversely related to the growth rate, and occurs most actively when cells enter the stationary phase. The levels of the three biosynthetic enzymes undergo corresponding changes under these conditions, suggesting that genetic control of enzyme biosynthesis may account for at least part of the regulation (Preiss, J., Annu. Rev. Microbiol. 38, 419-458 (1984)). In Escherichia coli, the structural genes for glycogen biosynthesis are clustered on adjacent operons—glgBX and glgCAP. Interestingly, the glycogen biosynthetic (glgCA) and degradative (glgP) genes are localized together in a cluster, possibly to facilitate the regulation of these systems in vivo (Romeo, T., Gene. 70(2), 363-76 (1988)). The glgC gene is the structural gene for glucose-1-phosphate adenylyltransferase. Synonyms for glucose-1-phosphate adenylyltransferase are ADP-glucose synthase, ADP-glucose pyrophosphorylase, ADP: a-D-glucose-1-phosphate adenylyltransferase, GlgC protein.
Glucose-1-phosphate adenylyltransferase (EC 2.7.7.27) is an allosteric enzyme in the glycogen biosynthetic pathway of eubacteria. Among the enteric bacteria, glucose-1-phosphate adenylyltransferase is regulated by glycolytic intermediates with fructose 1,6-biphosphate as the activator and AMP, ADP, and Pi as inhibitors. The enzyme catalyzes the synthesis of ADP glucose and PPi from glucose 1-phosphate and ATP. This reaction is the first unique step in bacterial glycogen biosynthesis.
It is known that the carbon storage regulatory system of Escherichia coli controls the expression of genes involved in carbohydrate metabolism and cell motility. CsrA binding to glgCAP transcripts inhibits glycogen metabolism by promoting glgCAP mRNA decay. CsrB RNA functions as an antagonist of CsrA by sequestering this protein and preventing its action (Baker, C. S. et al, Mol. Microbiol., 44(6), 1599-610 (2002)).
The glgCAP operon is under the positive control of cyclic AMP (cAMP) and the cAMP receptor protein (CRP). Both the cya gene encoding adenylate cyclase (EC 4.6.1.1) and the crp gene encoding CRP are required for optimal synthesis of glycogen (Fletterick, R. J. and Madsen, N. B., Annu. Rev. Biochem., 49, 31-61 (1980)). CRP binds to a site located upstream of the glgC gene. Glycogen synthesis in E. coli is also positively regulated by ppGpp, which stimulates the transcription of the glgCAP operon (Preiss. J., and Romeo, T., Prog. Nucleic Acid Res. Mol. Biol. 47, 299-329 (1994)).
By using a mini-Mu random chromosomal library and screening for glycogen overproduction, a novel gene (glgS) involved in glycogen synthesis was identified (Hengge-Aronis, R. and Fischer, D., Mol Microbiol. 6, 14, 1877-86 (1992)). It was also shown that the Escherichia coli protein GlgS is up-regulated in response to starvation stress and its overexpression was shown to stimulate glycogen synthesis (Kozlov, G. et al, BMC Biol., 2, 1, 10 (2004)).
The initial substrate for glycolysis biosynthesis is glucose-1-P, obtained from glucose-6-P, so said pathway competes with glycolysis for glucose-6-P. But currently, there have been no reports of inactivating the glgBX and/or glgCAP operons or inactivating the glgS gene for producing L-amino acids.